Plastidial-targeting peptide

ABSTRACT

The invention relates to a non-cleavable, plastidial targeting polypeptide derived from a protein from the inner membrane of plant chloroplasts. Said peptide is particularly suitable for importing proteins of interest in plasts.

The present invention relates to the production of proteins of interestin plants, and in particular to the targeting thereof to the plastidcompartment.

Plasts are intracellular organelle of chlorophyll-containing plants(algae, mosses, and higher plants). Several main types of plasts can bedistinguished according to their pigment content and the nature thereof:amyloplasts, which are rich in starch, chloroplasts in which the mainpigments are chlorophylls, and chromoplasts for which the main pigmentsare keratonoids. These three categories of plasts which derive fromcommon precursors, proplasts, have a common basic structure consistingof a double membrane enclosing the plastid stroma. In chloroplasts,there is a third membrane system forming, within the stroma, sacculescalled thylakoids.

Besides their essential role in photosynthesis, chloroplasts are alsoinvolved in redox reactions, for example the reduction of nitrates toammonium. Plasts also play an essential role in the biosynthesis and/orthe storage of many molecules, among which mention will be made ofstarch, lipids, carotenoids, most amino acids, plant hormones (abscissicacid, precursors of gibberellins, jasmonate, etc.).

Although plasts have their own genome encoding some of their proteins, alarge number of the enzymes involved in the various plastid functionsare encoded by the nuclear genome and imported into the plasts.

This importation is carried out via a specific mechanism, which has moreparticularly been studied in the case of chloroplasts (for review cf.CHEN and SCHNELL, Trends Cell Biol. 9, 222-227, 1999; KEEGSTRA andCLINE, The Plant Cell 11, 557-570, 1999; SCHLEIFF and SOLL, Planta 211,449-456, 2000; JACKSON-CONSTAN and KEEGSTRA, Plant Physiol. 125,1567-1676, 2001). This mechanism involves an import system in each ofthe two plastid membranes: in the outer membrane, the Toc (translocon atouter membrane of chloroplast) complex which comprises at least threeproteins: Toc 86, 75 and 34 (KESSLER et al., Science 266, 1035-1039,1994; PERRY and KEEGSTRA, Plant Cell 6, 93-105, 1994); in the innermembrane, the Tic (translocon at inner membrane of chloroplast) complexwhich comprises at least four proteins: Tic 110, 55, 22 and 20 (KESSLERand BLOBEL, Proc. Natl. Acad. Sci. 93, 7684-7689, 1996; LÜBECK et al.,EMBO J. 15, 4230-4238, 1996; CALIEBE et al., EMBO J. 16, 7342-7350,1997; KOURANOV et al., J. Cell Biol., 143, 991-1002, 1998), and also achaperone protein in the stroma: ClpC (AKITA et al., J. Cell Biol. 136,983-994, 1997; NIELSEN et al., EMBO J. 16, 935-946, 1997).

A major element of this mechanism is Toc75, which is the most abundantprotein in the outer membrane, and forms the central pore of thetranslocation channel located in this membrane (SCHNELL et al., Science266, 1007-1012, 1994; TRANEL et al., EMBO J. 14, 2436-2446, 1995). Toc75interacts specifically with a particular sequence, called “targetingpeptide” or “transit peptide”, located at the N-terminal end of theproteins imported into the plasts (MA et al., J. Cell Biol. 134,315-327, 1996).

Many targeting peptides have been identified in the precursors ofproteins targeted to the intermembrane space, the inner membrane, thestroma and, in the case of chloroplasts, to the thylakoid membrane.

Among the proteins known to have a cleavable intraplastid-targetingpeptide, mention will in particular be made of proteins targeted to theintermembrane space (Tic22: KOURANOV et al., 1998, mentioned above;KOURANOV et al., J. Biol. Chem. 274, 25181-25194, 1999), proteinstargeted to the inner membrane (TPT (Triose-Pi/Pi translocator): BRINKet al., J. Biol. Chem. 270, 20808-20815, 1995), proteins targeted to thestroma (ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit):DE CASTRO SILVA FILHO et al., Plant Mol. Biol. 30, 769-780, 1996;carbonic anhydrase), proteins targeted to the thylakoid membrane (LHCP(light harvesting complex): LAMPPA et al., J. Biol. Chem. 263,14996-14999, 1988; Cfo-II: ATPase subunit) and to the thylakoid lumen(OEE1 (Oxygen Evolving Element 1): KO and CASHMORE, EMBO J. 8,3187-3194, 1989).

These targeting peptides generally comprise between 40 and 100 aminoacids, and most of them have common characteristics: they are virtuallydevoid of negatively charged amino acids, such as aspartic acid,glutamic acid, asparagine or glutamine; their N-terminal region isdevoid of charged amino acids, and of amino acids such as glycine orproline; their central region contains a very high proportion of basicor hydroxylated amino acids, such as serine or threonine; theirC-terminal region is rich in arginine and has the ability to form anamphipathic, beta-sheet secondary structure.

In the case of proteins targeted to the thylakoid lumen, the targetingpeptide is bipartite and comprises additional information for crossingthe thylakoid membrane (DE BOER and WEISBEEK, Biochim. Biophys. Acta.1071, 221-253, 1991). In certain cases, this bipartite targeting peptidecan also be found in proteins targeted to the thylakoid membrane(KARNAUCHOV et al., J. Biol. Chem. 269, 32871-32878, 1994).

In all cases, the targeting peptide is cleaved after importation. Thiscleavage is carried out by specific proteases; a protease located in thestroma (VANDERVERE et al., Proc. Natl. Acad. Sci. 92, 7177-7181, 1995),and a protease located in the lumen of the thylakoid (CHAAL et al., J.Biol. Chem. 273, 689-692, 1998) have been described.

Proteins targeted to the outer membrane do not generally comprise acleavable signal peptide; the targeting information is contained in themature protein (CLINE and HENRY, Annu. Rev. Cell Dev. Biol., 12, 1-26,1996); after they have been synthesized in the cytosol, these proteinsare directly incorporated into the membrane (VAN'T HOF et al, FEBS lett.291, 350-354, 1991 and J. Biol. Chem. 268, 4037-4042, 1993; PINADUWAGEand BRUCE, J. Biol. Chem. 271, 32907-32915, 1996) by means ofinteractions, the nature of which remains poorly understood, with thelipid bilayer. The only known exception to date concerns the Toc75protein or (OEP75), the targeting of which to the outer membranerequires the presence of a cleavable, bipartite N-terminal targetingpeptide (TRANEL et al., 1995, mentioned above; TRANEL and KEEGSTRA,Plant Cell 8, 2093-2104, 1996).

It is known that the use of plast-targeting peptides is necessary forintroducing into these plasts proteins of interest for acting on variousplastid functions, in particular with the aim of improving thecharacteristics of plants of agronomic interest, for example thebiosynthesis of lipids, of starch, of vitamins, of hormones or ofproteins by said plants, or their resistance to diseases, to insects orto herbicides. For example, application EP 189707 proposes the use ofcleavable targeting peptides derived from chloroplast proteinprecursors, and in particular of the ribulose-1,5-bisphosphatecarboxylase small subunit targeting peptide, for importing a protein ofinterest into chloroplasts; PCT application WO 00/12732 proposes the useof targeting peptides from various plastid proteins, for importingproteins of interest into plasts.

The plastid functions can be modified in this way, and thecharacteristics conferred by these modifications are very diverse.

For the purposes of nonlimiting illustration, mention may be made of:

-   -   an increase in herbicide resistance, by expression of the        precursor of acetolactate synthetase (ALS), (LEE et al. EMBO J.,        7, 1241-1248, 1988), of mutated acetolactate synthetase (PRESTON        and POWLES, Heredity 88, 8-13, 2002); CHONG and CHOI, Biochem.        Biophys. Res. Commun. 279, 462-467, 2000), or of        3-enolpyruvylshikimate-5-phosphate synthetase (EPSP synthetase)        (KLEE et al., Mol. Gen. Genet., 210, 437-442, 1987);    -   an increase in resistance to various stresses, by expression of        zeaxanthin epoxidase, (SEQ et al., Trends Plant Sci., 7, 41-48,        2002), of choline monooxygenase (SHEN et al., Sheng Wu Gong        Cheng Xue Bao, 17, 1-6, 2001), of the product of the ERD1_ARATH        gene (KIYOSUE et al., Biochem. Biophys. Res. Commun., 15, 196,        1214-1220, 1993), of ferrochelatase (CHOW et al., J. Biol.        Chem., 31, 272, 27565-27571, 1997), of omega-3 fatty acid        desaturase (IBA et al., Tanpakushitsu Kakusan Koso, 39,        2803-2813, 1994; MURAKAMI et al.; Science 21, 287(5452),        476-479, 2000), or glutamine synthetase (FUENTES et al., J. Exp.        Bot., 52, 1071-1081, 2001);    -   modification of plast metabolism, so as to increase the capture        of light energy (GAUBIER et al., Mol. Gen. Genet., 1, 249,        58-64, 1995), the photosynthetic and growth capacities (MIYAGAWA        et al., Nature Biotech., 19, 965-969, 2001), the carotenoid        content (HUGUENEY et al., Eur. J. Biochem., 1, 209, 399-407,        1992; MANN et al., Nature Biotech., 18, 888-892, 2000), or the        content of various substances of interest, such as starch (PCT        application WO 00/11144), essential amino acids (MUEHLBAUER et        al., Plant Physiol., 106, 1303-1312, 1994), provitamin A (ROMER        et al., Nature Biotech., 18, 666-669, 2000), hormones (JOYARD et        al., Plant Physiol. 118, 715-723, 1998), etc.;    -   overexpression and chloroplast-targeting of proteins that can be        used for the purposes of bioremediation (detoxification or        depollution of contaminated soils), such as ferritin, (LOBREAUX        et al., Biochem. J., 15, 288(Pt 3), 931-939, 1992), proteins of        the phytochelatin family (CAZALE and CLEMENS, FEBS 507, 215-219,        2001; TSUJI et al., BBRC 293, 653-659, 2002), etc.

All the intraplastid-targeting peptides known in the prior art make itpossible to import a protein into plasts by means of the TOC and TICmembrane import systems, as indicated above. It has been noted that theuse of these peptides for targeting proteins of interest intochloroplasts may, in particular when the targeting peptide/protein ofinterest construct is placed under control of a strong promoter such asthe 35S promoter, has the drawback of saturating these import systems,by competing with the proteins naturally targeted to the chloroplast. Asa result of this, “leakages” occur, resulting, after a few days, in thepresence of the protein of interest in other subcellular compartmentssuch as the cytoplasm.

It would be desirable to have intraplastid-targeting peptides whichwould not depend on the TOC/TIC import system and would therefore makeit possible to avoid the abovementioned drawbacks.

In previous studies aimed at identifying, by means of a proteomicapproach, proteins from spinach chloroplast membrane preparations, theinventors identified, inter alia, peptides having considerable sequencesimilarity with a putative 41 kDa protein from Arabidopsis (TrEMBLaccession number Q9SV68) (SEIGNEURIN-BERNY et al., Plant. J. 19,217-228, 1999; FERRO et al., Electrophoresis 21, 3517-3526, 2000).

In continuing their studies in order to more fully characterize theArabidopsis protein, and its homologue in spinach, the inventors notedthat, surprisingly, although this involves proteins synthesized in thecytoplasm and imported at the inner membrane of the chloroplast, theimportation thereof was carried out without cleavage of a targetingpeptide; in addition, sequence analysis of these proteins reveals nosequence having the characteristics of known plastid-targeting peptides.

The proteins of the family represented by the 41 kDa Arabidopsisprotein, and the homologue spinach protein, are referred to hereinafteras IE41 (IE for Inner Envelope according to the nomenclatureconventionally used for this membrane system).

The sequence of the Arabidopsis IE41 protein is represented in thesequence listing in the appendix under the number SEQ ID NO: 1; thesequence of the cDNA encoding the spinach IE41 protein is represented inthe sequence listing in the appendix under the number SEQ ID NO: 2, andthe corresponding polypeptide sequence is represented in the sequencelisting in the appendix under the number SEQ ID NO: 3.

The inventors investigated which regions of the IE41 proteins wereinvolved in their plastid targeting, and identified a region of 41 aminoacids (residues at 60 to 100) that was essential to this targeting.

The sequence of this region is represented in the sequence listing inthe appendix under the number SEQ ID NO: 4 for the Arabidopsis IE41protein, and under the number SEQ ID NO: 5 for the spinach IE41 protein.

They also noted that, when fragments of IE41 containing this region werefused to the N-terminal end of a heterologous protein, the recombinantprotein resulting from this fusion was targeted to chloroplasts in amanner similar to the whole IE41 protein.

A subject of the present invention is an intraplastid-targetingpolypeptide, characterized in that it comprises:

-   -   a domain A consisting of a polypeptide having at least 60%,        preferably at least 70%, advantageously at least 80%, and        entirely preferably at least 90% identity, or at least 65%,        preferably at least 75%, advantageously at least 85%, and        entirely preferably at least 95% similarity, with one of the        polypeptides SEQ ID NO: 4 or SEQ ID NO: 5;    -   and at least one domain chosen from:    -   a domain B located at the N-terminal end of domain A, and        consisting of a fragment of one of the polypeptides SEQ ID NO: 1        or SEQ ID NO: 3 comprising at least amino acids 49 to 59,        preferably at least amino acids 39 to 59, advantageously at        least amino acids 29 to 59, entirely preferably at least amino        acids 19 to 59, and particularly advantageously at least amino        acids 9 to 59 of said polypeptide, or else of a polypeptide        having at least 60%, preferably at least 70%, advantageously at        least 80%, and entirely preferably at least 90% identity, or at        least 65%, preferably at least 75%, advantageously at least 85%,        and entirely preferably at least 95% similarity, with said        fragment;    -   a domain C located at the C-terminal end of domain A, and        consisting of a fragment of one of the polypeptides SEQ ID NO: 1        or SEQ ID NO: 3 comprising at least amino acids 101 to 111,        preferably at least amino acids 101 to 121, advantageously at        least amino acids 101 to 131, entirely preferably at least amino        acids 101 to 141, and particularly advantageously at least amino        acids 101 to 151 of said polypeptide, or else of a polypeptide        having at least 60%, preferably at least 70%, advantageously at        least 80%, and entirely preferably at least 90% identity, or at        least 65%, preferably at least 75%, advantageously at least 85%,        and entirely preferably at least 95% similarity, with said        fragment.

The percentage identities or the percentage similarities mentioned hereare determined by means of the BLASTp software (ALTSCHUL et al., NucleicAcids Res. 25, 3389-3402, 1997), using the default parameters.

Domains A, B and/or C defined above can come from the same IE41 protein;they can also come from IE41 proteins of different origins.

A subject of the present invention is also any chimeric polypeptideresulting from the fusion of an intraplastid-targeting polypeptide inaccordance with the invention with a heterologous polypeptide. Saidheterologous polypeptide may be any polypeptide of interest that it isdesired to introduce into plasts. Preferably, the intraplastid-targetingpolypeptide in accordance with the invention is placed at the N-terminalend of the heterologous peptide. It could, however, also be placedwithin this peptide, or else at its C-terminal end.

A subject of the present invention is also the use of anintraplastid-targeting polypeptide in accordance with the invention, forthe importation of a protein of interest into plasts, and advantageouslyfor the targeting of said protein to the inner plastid membrane.

According to a preferred embodiment of the present invention, saidintraplastid-targeting polypeptide is used for the importation of saidprotein of interest into chloroplasts.

In particular, a subject of the present invention is a method forimporting a protein of interest into plasts, characterized in that itcomprises the expression, in a plant cell containing said plasts, of achimeric polypeptide resulting from the fusion of anintraplastid-targeting polypeptide in accordance with the invention,with a heterologous polypeptide.

A subject of the present invention is also:

-   -   any polynucleotide encoding an intraplastid-targeting        polypeptide or a chimeric polypeptide in accordance with the        invention;    -   any recombinant expression cassette comprising a polynucleotide        in accordance with the invention placed under the control of        suitable sequences for regulating the transcription (in        particular transcription promoter and terminator);    -   any recombinant vector resulting from the insertion, into a        suitable host vector, of a polynucleotide or of an expression        cassette in accordance with the invention.

A subject of the present invention is also host cells harboring apolynucleotide, an expression cassette or a recombinant vector inaccordance with the invention.

The present invention also encompasses transgenic plants geneticallytransformed with a polynucleotide or an expression cassette inaccordance with the invention, and also the progenies of these plants.The invention also comprises the plant cells and tissues, and also theorgans or parts of plants, including leaves, stems, roots, flowers,fruits and/or seeds obtained from these plants.

Conventional techniques for constructing recombinant vectors, fortransforming host cells or organisms, and for producing recombinantproteins can be used for implementing the present invention.

The choice of the host vector and of the sequences for regulating theexpression will be made in particular according to the method oftransformation and to the host plant chosen, and/or to the type of cellor of tissue in which it is desired to obtain the expression.

A very large number of promoters that can be used for the expression inplant cells are known in themselves. By way of examples, a constitutivepromoter, such as the CaMV ³⁵S promoter or its derivatives, or thepromoter of actin or ubiquitin, etc., may be chosen. An induciblepromoter or else a tissue-specific promoter may also be chosen, so as toeffect the plastid-targeting of the protein of interest only at certainstages of the plant's development, under certain environmentalconditions, or in certain target tissues.

For example, if it is desired to preferentially obtain targeting of theprotein of interest to chloroplasts, a chimeric polypeptide inaccordance with the invention will be expressed under the control of apromoter specific for tissues or organs that are rich in plasts. By wayof examples, the promoters of the chlorella virus that regulateexpression of the adenine methyltransferase gene (MITRA and HIGGINS,Plant Mol. Biol. 26, 85-93, 1994) or that of the cassaya mosaic virus(VERDAGUER et al., Plant Mol. Biol. 37, 1055-1067, 1998) are expressedmainly in the green tissues. The regulatory elements of the promoter ofthe tomato 2A11 gene allow specific expression in the fruit (VAN HAARENand HOUCK, Plant Mol. Biol. 17, 615-630, 1991), etc.

Methods for transforming plant cells or whole plants are well known inthemselves: by way of nonlimiting examples, mention will be made of thetransformation of protoplasts in the presence of polyethylene glycol,electroporation, the use of a particle gun, cytoplasmic or nuclearmicroinjection, or transformation by means of Agrobacterium.

The present invention can be implemented in the usual applications ofplast-targeting peptides, and in particular in those mentioned above, soas to act on various plastid functions. This involves, in particular,the modification of functions specific to the inner membrane of theenvelope, for example the biosynthesis of pigments, of quinones, offatty acids, of vitamins and of plant hormone precursors, but also theimportation of all the ions and metabolites into the plast.

The characteristics of the plastid-targeting peptides in accordance withthe invention, which are very different from those of the knownplastid-targeting peptides, make it possible to suppose that thetargeting peptides in accordance with the invention use an import systemthat is different from that involving the TOC and TIC proteins.

As a result of this, the proteins of interest targeted to plasts bymeans of a targeting peptide in accordance with the invention would notcompete with the proteins naturally targeted to the plast by means ofthe TOC and TIC system, and would not saturate the latter. This wouldmake it possible in particular to prevent leakages into the othersubcellular compartments, and to conserve the proteins of interest inthe chloroplast, even after several days of expression. This would alsomake it possible to target to the plasts proteins for which conventionalimportation by means of an N-terminal cleavable targeting sequence andusing the TIC/TOC system would not be functional.

The present invention will be understood more fully from the furtherdescription which will follow, which refers to nonlimiting examplesillustrating the obtaining and characterization of plastid-targetingpeptides in accordance with the invention and their use for theimportation of heterologous proteins into chloroplasts.

EXAMPLE 1 Characterization and Cloning of the Arabidopsis thaliana IE41Protein

In prior studies aimed at identifying the most hydrophobic proteins ofspinach chloroplast membrane preparations (SEIGNEURIN-BERNY et al.,Plant. J., 19, p. 217-228, 1999; FERRO et al., Electrophoresis, 21,3517-3526, 2000), several peptides derived from a 41 kDa proteininhibiting great sequence similarity with a putative protein fromArabidopsis (TrEMBL accession number Q9SV68) were demonstrated.

However, analysis of the primary sequence of this 41 kDa protein bymeans of the TMPred program (HOFMANN and STOFFEL, Biol. Chem.Hoppe-Seyler., 347, 166, 1993), did not make it possible to detecttransmembrane segments able to provide anchoring of the protein in alipid bilayer.

To confirm the location of this protein in the chloroplast envelope, thecorresponding cDNA was cloned and the recombinant protein wasoverexpressed in E. coli in order to obtain polyclonal antibodiesdirected against this protein.

Expression in E. coli

The cDNA encoding the 41 kDa Arabidopsis protein is obtained by PCR,from an Arabidopsis cDNA library, using the following primers:

-   -   TCACATATGGCTGGAAAACTCAATGCAC (SEQ ID NO: 10)    -   which makes it possible to introduce an NdeI restriction site        (underlined) at the 5′ end of the cDNA;    -   ATGGATCCAACGCTCTTATGGCTCGAC (SEQ ID NO: 11)    -   which makes it possible to introduce a BamHI restriction site        (underlined) at the 3′ end of the cDNA.

The amplification fragment is cloned into the plasmid pBluescript KS⁻.The insert is then digested with the NdeI and BamHI restriction enzymes,and inserted into the expression vector pET-15b (NOVAGEN).

The resulting vector allows the expression of a recombinant proteinhaving a polyhistidine extension (His-tag) at its N-terminal end[(His-tag)-P41].

This vector is used for transforming E. coli strain BLR(DE3) bacteria.

The recombinant bacteria are cultured in 500 ml of LB medium containing100 μg/ml of ampicillin at 37° C. When the optical density at 600 nm(DO₆₀₀) of the E. coli culture reaches 0.6, IPTG(isopropyl-β-D-galactothiopyranoside) is added at a final concentrationof 1 mM so as to induce expression of the 41 kDa protein.

After 3 hours of culture, the cells are centrifuged for 2 min at 13 000rpm (EPPENDORF 5415D).

The pellet is resuspended in 20 ml of lysis buffer (50 mM NaH₂PO₄, 300mM Na Cl, 10 mM imidazole, pH 8) and the bacteria are lysed bysonication (OMRON sonicator, type STP.YM.220.VAC, 6×1 min, 0° C.).

After sonication, the total protein extract is analyzed by SDS 12% PAGE.The gels are stained with Coomassie blue (R-250, BIORAD).

The results are given in FIG. 1A:

Legend of FIG. 1A:

-   pET-15b=bacteria transformed with the nonrecombinant plasmid    (negative control)-   pET-15b+insert=bacteria transformed with the recombinant plasmid,-   −=no induction with IPTG,-   +=induction with IPTG,-   *=band corresponding to the recombinant 41 kDa protein.

After induction with IPTG, the recombinant protein is strongly expressedin the bacteria transformed with the plasmid pET-15b containing theArabidopsis cDNA insert.

Solubilization of the Recombinant Protein

The bacterial pellet is suspended in 1 ml of 20 mM Tris/HCl buffer, pH6.8, and then lysed by sonication (OMRON sonicator, type STP.YM.220.VAC,6×1 min, 0° C.).

After sonication, a first centrifugation (2 min, 13 000 rpm) of thetotal protein extract makes it possible to isolate the soluble proteinsin the supernatant. The insoluble proteins in the pellet are suspendedin 1 ml of a buffer containing detergent (20 mM Tris/HCl pH 6.8, 0.5%Triton X-100). A second centrifugation makes it possible to isolate themembrane proteins solubilized with Triton X-100. The nonsolubilizedproteins are resuspended in 20 mM Tris/HCl, pH 6.8 and analyzed. Thevarious fractions are analyzed by SDS-12% PAGE, as indicated above.

The results are given in FIG. 1B:

Legend of FIG. 1B:

-   pET-15b=bacteria transformed with the nonrecombinant plasmid    (negative control),-   pET-15b+insert=bacteria transformed with the recombinant plasmid,-   S=soluble proteins,-   D=proteins solubilized in Triton X-100,-   I=proteins not solubilized in Triton X-100,-   *=band corresponding to the recombinant 41 kDa protein.

It is noted that the recombinant protein (*) is found in 2 differentfractions: a water-soluble fraction present in the E. coli cytosol andan insoluble fraction, which is not solubilized by Triton X-100, whichindicates that it is probably aggregated in the form of inclusionbodies.

Purification of the Recombinant Protein

The soluble fraction of the recombinant (His-tag)-P41 protein ispurified by affinity chromatography. After centrifugation (10 min at6000 rpm, EPPENDORF 5415D), the supernatant is loaded onto a 2.5 ml“His-bind resin” affinity column (NOVAGEN) charged with 13 ml of chargedbuffer (50 mM NiSO₄) and equilibrated with 5 ml of equilibrating buffer(20 mM tris/HCl, pH 7.9, 5 mM imidazole, 0.5 M NaCl).

The column is washed with 2 volumes containing 35 mM imidazole, and 2volumes of lysis buffer containing 60 mM imidazole (L2). The protein iseluted with 6 volumes of lysis buffer containing 250 mM imidazole. Thevarious fractions are analyzed by SDS-12% PAGE and revealed withCoomassie blue.

The results are given in FIG. 2.

Legend of FIG. 2:

-   C=bacterial pellet diluted in the lysis buffer (10 μl),-   S=soluble bacterial proteins (10 μl),-   P=proteins not bound (10 μl),-   L, L1, L2=washes with the lysis buffer (35 and 60 mM imidazole) (15    μl),-   Elution=fraction eluted in the presence of 250 mM imidazole.    Production of Polyclonal Antibodies

The purified recombinant (His-tag)-P41 protein is desalified (SEPHADEXG25 column) and stored at −80° C.

This recombinant protein is used for immunizing rabbits in order toproduce polyclonal antibodies directed against the 41 kDa Arabidopsisprotein.

EXAMPLE 2 Location of the 41 kDa Protein in Chloroplasts

During the purification procedure, the 41 kDa protein behaves like aprotein that is water soluble and slightly hydrophobic, which raises thequestion of its effective association with the chloroplast envelope.

Its subcellular location was therefore verified by analyzing variouschloroplast fractions.

Crude chloroplasts are obtained from 3-4 kg of spinach (Spinaciaoleracea L.) leaves and are purified by isopycnic centrifugation on aPercoll gradient (DOUCE and JOYARD, Methods in chloroplast MolecularBiology. Edelman, M., Hallick, R. and Chua, N.-H., eds. (Amsterdam:Elsevier Science Publishers BV), 239-256, 1982). At this stage, proteaseinhibitors (1 mM PMSF, 1 mM benzamidine and 0.5 mM aminocaproic acid)are added in order to prevent any protein degradation. The purifiedchloroplasts are lysed in a hypotonic medium, and the envelope membranesare purified from the lysate by centrifugation on a sucrose gradient(DOUCE and JOYARD, 1982, mentioned above). Envelope subfractionsrespectively enriched in outer and inner membranes are obtainedaccording to the protocol described by BLOCK et al. (J. Biol. Chem.,258, 13273-13280, 1983).

All the above steps are carried out at between 0 and 5° C. The fractionsobtained are stored in liquid nitrogen in 50 mM MOPS-NaOH, pH 7.8, inthe presence of protease inhibitors (1 mM benzamidine and 0.5 mMaminocaproic acid).

Analysis of the Chloroplast Fractions

The SDS-PAGE analyses of the total chloroplasts, or of their envelopemembrane, stroma or thylakoid membrane fractions (15 μg), and also ofthe chloroplast envelope subfractions (15 μg), are carried out asdescribed by CHUA (Methods Enzymol., 69, 434-436, 1980). The resolvingand stacking gels (12-15% acrylamide), like the migration buffer,contain 0.1% of SDS. The peptides are revealed either with Coomassieblue (MANIATIS et al., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1982) or with silver nitrate (MERRIL et al., Anal.Biochem., 110, 201-207, 1981).

For the Western blotting analyses, the 41 kDa protein is detected usingthe polyclonal antibodies directed against the recombinant Arabidopsisprotein produced as described in example 1, labeled with alkalinephosphatase.

The results are given in FIG. 3A.

Legend of FIG. 3A:

-   C=chloroplast proteins,-   E=envelope membrane proteins,-   S=stroma proteins,-   T=thylakoid membrane proteins.

These results show that the 41 kDa protein is associated only with thechloroplast envelope and is not detected in the stroma or in thethylakoids.

The purified intact chloroplasts are devoid of cytosolic proteins whichcan contaminate the preparation (DOUCE and JOYARD, 1980, mentionedabove). However, the 41 kDa protein could interact specifically with theouter membrane of the chloroplast envelope and could thus be co-purifiedwith the envelope preparations. In order to exclude this possibility, 20μg of envelope derived from intact chloroplasts are treated withBacillus thermoproteolyticus thermolysin (0, 20, 50 and 100 μg/ml) inorder to digest the polypeptides located on the outer surface of theouter membrane of the envelope (JOYARD et al., J. Biol. Chem., 258, 10000-10 006, 1983). As a control, solubilized envelope proteins aresubjected to the same proteolytic treatment.

The presence of the 41 kDa protein is detected by Western blotting, asdescribed above.

The results are given in FIGS. 3B and 3C.

Legend of FIGS. 3B and 3C

-   0=absence of thermolysin,-   20=thermolysin at 20 μg/ml,-   50=thermolysin at 50 μg/ml,-   100=thermolysin at 100 μg/ml.

The 41 kDa protein is not affected by the treatment with thermolysin(FIG. 3B), whereas the same proteolytic treatment carried out onsolubilized envelope proteins shows the sensitivity of the 41 kDaprotein to the thermolysin treatment (FIG. 3C). This result excludes thehypothesis that the 41 kDa protein is located on the outer face of theouter membrane. In fact, the 41 kDa protein is not a cytosolic proteincontaminating the chloroplast envelope preparations.

Envelope subfractions respectively enriched in outer and inner membranesare used to specify the sub-location of the 41 kDa protein at the levelof the membrane of the chloroplast envelope. The nature of theseenvelope subfractions was confirmed using the markers IE18 and OEP24,which are respectively intrinsic proteins of the inner and outermembrane of the chloroplast envelope. The IE18 and OEP24 proteins aredetected using polyclonal antibodies directed specifically against eachof these proteins.

The results are given in FIG. 3D.

Legend of FIG. 3D:

-   E=envelope membrane proteins,-   OM=outer membrane proteins,-   IM=inner membrane proteins.

The 41 kDa protein is found, like the IE18 protein, only in thepreparations of chloroplast envelopes enriched in inner membrane.

All the results given in FIGS. 3A-3D show that the 41 kDa protein islocated at the level of the inner membrane of the chloroplast envelope.

According to conventional nomenclature, this inner envelope protein,which has a theoretical mass by polyacrylamide gel electrophoresis of 41kDa, is called IE41 (for “Inner Envelope Protein of 41 kDa”).

Analysis of the Interactions Between the IE41 Protein and the InnerMembrane of the Chloroplast Envelope

In order to analyze more precisely the mode of interaction of the IE41protein with the chloroplasts inner membrane, various hypotheses weretested:

1) The IE41 protein is a soluble protein located in the intermembranespace between the outer and inner membranes of the chloroplast envelope.This protein would be co-purified with the envelope preparations andsequested in the envelope vesicules. In this case, sonication of theenvelope vesicules would make it possible to release IE41.

To test this first hypothesis, envelope proteins (500 μg) aresolubilized in 50 mM MOPS, pH 7.8 (500 μl). The envelope vesicules aresonicated for 10 sec and then centrifuged (20 min at 72 000 g, BeckmanL2 65B, SW28 rotor) in order to separate the soluble proteins and themembrane proteins. Each fraction (20 μl) is analyzed by SDS-12% PAGE(visualization: Coomassie blue) and Western blotting.

The results are given in FIG. 4A.

Legend of FIG. 4A:

-   −=no sonication,-   +=sonication,-   S=soluble proteins,-   I=membrane proteins.

The analyses by SDS-PAGE and Western blotting of the treated envelopefractions show that the IE41 protein is not solubilized after sonicationof the envelope vesicules. On the contrary, the major soluble proteinsof the stroma (RbcL) which are sequested in the envelope vesicules, andwhich are known to contaminate the envelope fractions, are solubilizedby this treatment. This shows that the IE41 protein is neither a solubleprotein of the intermembrane space, nor a soluble protein of the stromathat contaminates the purified envelope fraction.

2) The IE41 protein may be bound to the inner membrane:

-   -   either by anchoring to the lipid bilayer or partial insertion        into said bilayer; in this case, only the use of detergent can        enable the IE41 protein to be solubilized;    -   by electrostatic interactions with one or more membrane proteins        or the polar surface of the lipid bilayer; in this case, these        interactions can be broken, and the IE41 protein can be        solubilized by means of an alkaline treatment or by means of        high salt concentrations.

In order to determine the type of interactions involved in the bindingof IE41 with the inner membrane, the following experiments were carriedout:

a) Solubilization with Triton X-100

Envelope vesicules (0.8 mg) are diluted in 1 ml of 50 mM MOPS, pH 7.8,containing 0.05, 0.1 or 0.2% (v/v) of Triton X-100. After incubation for30 min at 4° C., the mixture is centrifuged in order to separate thesoluble proteins and the membrane proteins. All the fractions (20 μl)are analyzed by SDS-12% PAGE (visualization: Coomassie blue) and Westernblotting.

The results are given in FIG. 4C.

Legend of FIG. 4C:

-   Mix=purified envelope vesicules,-   M=envelope membrane proteins,-   S=envelope soluble proteins.

These results show that the IE41 protein can be completely solubilizedwith concentrations of Triton X-100 (0.2%) that are much lower thanthose (of the order of 2%) which are necessary for solubilizing theintrinsic proteins.

b) Solubilization by Alkaline Treatment or by Salt Treatment.

Purified envelope vesicules (500 μg) are incubated for 30 min at 4° C.in various media (500 μl):

-   -   1) 10 mM MOPS, pH 7.8+0.5 M NaCl;    -   2) 10 nM MOPS, pH 7.8+0.5 M KI;    -   3) 0.1 M Na₂CO₃, pH 11;    -   4) 0.1 N NaOH,    -   and then sonicated and centrifuged as described above in order        to separate the soluble proteins and the membrane proteins.

All the fractions are analyzed (20 μl) by SDS-12% PAGE (visualization:Coomassie blue) and Western blotting (detection with the polyclonalantibodies directed against the Arabidopsis IE41 protein (1/5000dilution) or directed against the IE18 protein (1/5000 dilution)).

The results are given in FIG. 4B

Legend of FIG. 4B:

-   +=sonication (10 sec),-   NaCl 0.5 M=treatment 1,-   KI 0.5 M=treatment 2,-   0.1 M Na₂CO₃, pH 11=treatment 3,-   0.1 N NaOH=treatment 4,-   S=soluble protein fraction,-   I=insoluble protein fraction.

These results show that the IE41 protein is at least partly solubilizedby the salt (KI, NaCl) or moderately alkaline (Na₂CO₃) treatments, whichhave no effect on the intrinsic protein IE18, it only being possible forthe latter to be solubilized by a strong alkaline treatment (NaOH).

All the results given in FIGS. 4A, 4B and 4C indicate that the IE41protein is an extrinsic protein, the binding of which to the innermembrane of the chloroplast envelope involves electrostatic interaction.

EXAMPLE 3 Purification and Characterization of the Spinach IE41 Protein

Surprisingly, the IE41 protein purified from spinach chloroplasts andthe recombinant Arabidopsis protein have a similar size by SDS-PAGE andWestern blotting, which suggest the possibility that IE41 may betargeted to the inner membrane of the envelope without requiring thecleavage of an N-terminal import sequence.

To test this hypothesis, the IE41 protein present in the envelope ofspinach chloroplasts was purified in order to sequence it and to compareits sequence with that of the corresponding cDNA.

Immunopurification of the Spinach IE41 Protein

Chloroplast envelope proteins (1 mg) are solubilized in 1 ml of 50 mMTris/HCl buffer containing 150 mM NaCl and 6 mM CHAPS and centrifuged(20 min, 72 000 g, Beckman L2 65B, SW28 rotor). The soluble proteins areincubated for 1 h at 4° C. with 33 μl of polyclonal serum directedagainst the recombinant Arabidopsis IE41 protein. 50 μg ofagarose-protein A (BOEHRINGER) are then added and the mixture isincubated for 3 h at 4° C. After 3 successive washes by centrifugation(EPPENDORF 5415D, 12 000 rpm, 20 min, 4° C.) and resuspension of thepellet in 1 ml of solubilizing buffer (20 mM MOPS, 150 mM NaCl, 6 mMCHAPS, pH 7.8), an excess (50 μg) of recombinant protein (His-tag)-IE41,in 200 μl of solubilizing buffer, is added. The mixture is incubated for1 h at 4° C., and centrifuged for 20 min at 12 000 rpm (EPPENDORF5415D). The supernatant is incubated for 1 h with Ni-NTA resin (QIAGEN),pre-equilibrated in the solubilizing buffer, in order to eliminate mostof the (His-tag)-IE41 recombinant protein.

Each fraction (20 μl) is analyzed by SDS-12% PAGE (visualization withsilver nitrate) and Western blotting (using the rabbit anti-IE41polyclonal antibodies described in example 1).

The results are given in FIG. 5.

Legend of FIG. 5:

-   A: analysis by SDS-PAGE;-   B: Western blotting;-   Mix=solubilized envelope proteins;-   C=insoluble proteins;-   S=soluble proteins;-   L1, L2, L3=fractions recovered in the course of the 3 successive    washes;-   E1=fraction eliminated by incubation with the Ni-NTA resin;-   E2=purified spinach IE41 protein (So)+(His-tag)-IE41.

The E2 fraction comprising the purified natural spinach IE41 protein(So) remains contaminated by the recombinant Arabidopsis (His-tag)-IE41protein.

The difference in size between these two proteins corresponds to thepolyhistidine extension (His-tag) present at the N-terminal end of therecombinant protein.

EXAMPLE 4 Obtaining the cDNA Encoding the Spinach IE41 Protein

Partial sequencing of the spinach IE41 protein eluted from theelectrophoresis gel made it possible to obtain 9 different peptidesequences. These sequences were used to define degenerate primers whichmade it possible to isolate the cDNA encoding the IE41 protein.

The complete sequence of this cDNA (SEQ ID No: 2), and also the deducedpolypeptide sequence (SEQ ID NO: 3), are given in FIG. 6. Thetranslation initiation codon ATG is indicated in bold letters, and thestop codon TAA is underlined. The 9 peptide sequences obtained by directsequencing of the spinach IE41 protein are highlighted in gray. Thecorrespondence between the peptides obtained with the sequence deducedfrom the cDNA of the spinach IE41 protein, in particular in theN-terminal region, and also the presence of a stop codon downstream ofthe initiating methionine and in the same reading frame, demonstratethat the predicted cDNA is complete and that this protein does notundergo any post-translational maturation during its targeting to theinner membrane of the chloroplast envelope.

The spinach IE41 protein has 75.1% identity and 88.8% similarity withthe Arabidopsis IE41 protein. This high similarity, and the fact thatArabidopsis contains only one ie41 gene per haploid genome, make itpossible to conclude that these proteins are encoded by orthologousArabidopsis and spinach genes.

The Arabidopsis and spinach IE41 proteins were aligned with homologousproteins from a bacterium, from a yeast and from animals.

The results are given in FIG. 7.

-   Arabidopsis thaliana: IE41 ATH (SEQ ID NO: 1),-   Spinach: IE41 SOL (SEQ ID NO: 3);

homologous proteins: Esherichia coli: QORECOLI, (SEQ ID NO: 6)Saccharomyces cerevisiae: QORYEAST, (SEQ ID NO: 7) Cavia Porcellus:QORCAVPO, (SEQ ID NO: 8) Mouse: QORMOUSE. (SEQ ID NO: 9)The residues conserved in the 6 peptide sequences are highlighted indark gray. The residues conserved in the IE41 sequence and in at leastone other homologous protein sequence are highlighted in light gray. Thesimilarities between residues are based on the following groups: ASPTG,ILMV, KRH, NQ, DE, YWF and C.

The homology searches indicate that the IE41 protein belongs to thedehydrogenase super family, and more particularly to the group ofξ-crystalline-type quinone oxidoreductases (JÖRNVALL et al., FEBS 3,240-244, 1993). In addition, the sequence comparison between the IE41proteins and the other proteins of the same family reveals that thefirst 50 residues in the N-terminal region of these proteins are veryconserved between bacteria, plants and animals. This observationsuggests that this N-terminal region of the plant IE41 proteins is notinvolved in targeting into the chloroplast, and is more probablyconserved due to the selection pressure exerted during evolution on thecatalytic domain of the protein.

EXAMPLE 5 Analysis of the Plastid-Targeting of the Arabidopsis IE41Protein in Arabidopsis and Tobacco Cells

In order to define the domain essential to the importation of the IE41protein, various constructs encoding truncated forms of this protein,fused to GFP, are expressed in Arabidopsis and tobacco cells.

Construction of the Expression Vectors:

The plasmid [35Ω-sGFP(S65T)] used for these constructs, which comprisesthe sequence encoding GFP under the control of the 35S promoter, andalso the plasmid [35Ω-TP-sGFP(S65T)], which comprises the sequenceencoding the targeting peptide (TP) of the ribulose-1,5-bisphosphatecarboxylase small subunit, fused to the sequence encoding GFP, werepreviously described by CHIU et al. (Curr. Biol., 6, 325-330, 1996).

The sequence encoding the Arabidopsis IE41 protein is amplified by PCRusing the following two primers: XhoI-N-terCCTCTCGAGATGGCTGGAAAACTCATGCAC, (SEQ ID NO: 12) and NcoI-C-terCAACCCATGGATGGCTCGACAATGATCTTC, (SEQ ID NO: 13)which introduce, respectively, an XhoI site and an NcoI site(underlined).

The PCR product is cloned, blunt-ended, into the vector pBLUESCRIPT KS(STRATAGENE). The XhoI-NcoI fragment cleaved from the plasmid thusobtained is inserted into the plasmid 35Ω-sGFP(S65T) digested beforehandwith SalI-NcoI, in order to create the vector 35Ω-IE41-sGFP(S65T),comprising the coding region of the Arabidopsis IE41 protein, fused toGFP. A similar protocol is used for the other constructs:

The sequence encoding the Arabidopsis IE41 protein lacking the first 31amino acids is obtained by PCR amplification using the following twoprimers: SalI-N-ter CGGTTGTCGACATGAAGAGTAATGAGGTTTGCCTG (SEQ ID NO: 14)NcoI-C-ter CAACCCATGGATGGCTCGACAATGATCTTC. (SEQ ID NO: 13)

The plasmid 35Ω-Δ(1-31)IE41-sGFP(S65T) is obtained by insertion of thissequence into the plasmid 35Ω-sGFP(S65T).

The sequence encoding the Arabidopsis IE41 protein lacking the first 59amino acids is amplified by PCR using the following two primers:SalI-N-ter GAATGGTCGACATGTTTCTGCCCCGCAAGTTC, (SEQ ID NO: 15) andNcoI-C-ter CAACCCATGGATGGCTCGACAATGATCTTC. (SEQ ID NO: 13)

The plasmid 35Ω-Δ(1-59)IE41-sGFP(S65T) is obtained by insertion of thissequence into the plasmid 35Ω-sGFP(S65T).

The sequence encoding the Arabidopsis IE41 protein lacking the first 99amino acids is amplified by PCR using the following two primers:SalI-N-ter GGTTGTCGACATGCTAGGTGGAGGTGGACTTG (SEQ ID NO: 16) NcoI-C-terCAACCCATGGATGGCTCGACAATGATCTTC. (SEQ ID NO: 13)

The plasmid 35Ω-Δ(1-99)IE41-sGFP(S65T) is obtained by insertion of thissequence into the plasmid 35Ω-sGFP(S65T).

The sequence encoding the amino acids 6-100 of the Arabidopsis IE41protein is amplified by PCR using the following two primers: XhoI-N-terCCTCTCGAGATGGCTGGAAAAACTCATGCAC (SEQ ID NO: 17) NcoI-C-terACCCATGGCTAGATGGCTAAGAACCGCTAC. (SEQ ID NO: 18)

The primer SEQ ID NO: 17 comprises an additional nucleotide comparedwith the primer SEQ ID NO: 15, which creates a reading frame shift inthe amplification product, the translation of which begins at the ATGcodon corresponding to the methionine at position 6 of the IE41 protein.

The plasmid 35Ω-(6-100)IE41-sGFP(S65T) is obtained by insertion of thissequence into the plasmid 35Ω-sGFP(S65T).

The sequence encoding amino acids 60-100 of the Arabidopsis IE41 proteinis amplified by PCR using the following two primers: SalI-N-terGAATGGTCGACATGTTTCTGCCCCGCAAGTTC (SEQ ID NO: 15) NcoI-C-terACCCATGGCTAGATGGCTAAGAACCGCTAC. (SEQ ID NO: 18)The plasmid 35Ω-(60-100)IE41-sGFP(S65T) is obtained by insertion of thissequence into the plasmid 35Ω-sGFP(S65T).

These various constructs are given in FIG. 8.

Legend of FIG. 8:

-   IE41=plasmid 35Ω-IE41-sGFP(S65T)-   Δ(1-31)IE41=plasmid 35Ω-Δ(1-31)IE41-sGFP(S65T)-   Δ(1-59)IE41=plasmid 35Ω-Δ(1-59)IE41-sGFP(S65T)-   Δ(1-99)IE41=plasmid 35Ω-Δ(1-99)IE41-sGFP(S65T)-   (6-100)IE41=plasmid 35Ω-(6-100)IE41-sGFP(S65T)-   (60-100)IE41=plasmid 35Ω-(60-100)IE41-sGFP(S65T)    Bombardment of Arabidopsis and Tobacco Cells

The Arabidopsis cells are cultured in light for 3 days in GAMBORG's B5medium (SIGMA, pH 5.8) supplemented with 1.5% sucrose and 1 μM ANA(naphthaleneacetic acid). 15 ml of cell suspension (corresponding toapproximately 0.5 g) are transferred into Petri dishes containing thesame growth medium to which 0.8% bacto-agar has been added, andincubated for 18-36 h in the light.

BY2 tobacco cells are cultured for 5 days at 27° C. in MURASHIGE andSKOOG medium (MS medium, DUCHEFA, pH 5.8) supplemented with 3% sucrose,0.2% KH₂PO₄, 0.2% myoinositol, 1 μM 2.4D (2.4-dichlorophenoxyaceticacid) and 3 μM thiamine. 2.5 ml of cell suspension (corresponding toapproximately 0.3 g) are transferred into Petri dishes containing thesame growth medium to which 1% bacto-agar has been added, and are placedat 27° C. for 18-24 h.

The plasmids comprising the test constructs used for the tissuebombardment are prepared using the “QIAfilter Plasmid Midi Kit” (Qiagen,Germany).

The plasmid [35Ω-sGFP(S65T)] (GFP) and the plasmid [35Ω-TP-sGFP(S65T)](TP-GFP) are respectively used as a negative control and as a positivecontrol.

The plasmids (1 μg) are introduced into the cells using a pneumaticparticle gun (PDS-1000/He, BIORAD). The bombardment conditions are asfollows: helium pressure of 1350 psi; 1100 psi rupture disks (BIORAD);10 cm target distance; 1 μm gold microcarriers (BIORAD) are used. Afterbombardment, the cells are incubated on these same Petri dishes for18-36 h (in the light for the Arabidopsis cells), and then transferredonto glass slides before fluorescence microscopy.

Fluorescence Microscopy

The location of the GFP and of the GFP-fusion peptides in analyzed inthe transformed cells by fluorescence microscopy using a ZEISS AXIOPLAN2 fluorescence microscope and a digital CCD camera (HAMAMATSU). The setsof filters used are: Zeiss filterset 13, 488013-0000 (exciter BP 470/20,beam splitter FT 493, emitter BP 505-530), and Zeiss filter set 15,488015-0000 (exciter BP 546/12, beam splitter FT 580, emitter LP 590)for the GFP and the chlorophyll autofluorescence, respectively.

Under these conditions, the presence of chlorophyll (specificallylocated in the chloroplasts) and the location of the GFP in the cell arevisualized by virtue of an intense fluorescence.

In the Arabidopsis cells transformed with the constructs GFP,Δ(1-99)IE41, and (60-100)IE41, the GFP fluorescence appears to bediffuse and located in the cytosol and the nucleus; no co-localizationwith chlorophyll is observed.

In the Arabidopsis cells transformed with the constructs IE41,Δ(1-31)IE41, Δ(1-59)IE41 and (6-100)IE41, and also with the positivecontrol for localization TP-GFP, a co-localization is, on the other handobserved in the chloroplasts, between the GFP fluorescence and thechlorophyll autofluorescence.

The results are similar in the nonchlorophyll-containing BY2 tobaccocells: the fluorescent labelings observed with the constructs IE41,Δ(1-59)IE41 and (6-100)IE41, and with the positive control forlocalization TP-GFP, correspond to a plastid localization; on the otherhand, the fluorescent labelings observed with the constructs GFP,Δ(1-99)IE41, and (60-100)IE41 correspond to a cytosolic and nuclearlocalization.

These experiments show that the targeting is also effective innonchlorophyll-containing plasts.

All the results above show that:

-   -   the complete IE41 protein fused to GFP is targeted into the        chloroplast;    -   the 59 residues located at the N-terminal are not essential to        the importation;    -   the 99 residues located at the N-terminal contain a region        essential to the importation;    -   a sequence of 94 residues, corresponding to N-terminal amino        acids 6-100, is sufficient to catalyze the importation; the 223        C-terminal residues (101-323) are therefore not essential to the        importation.

The internal sequence of 40 amino acids, ranging from residues 60-100,correspond to the domain that is essential for importation. However,this domain, which must be present in order to direct the protein to theplasts, is not sufficient for correct targeting.

EXAMPLE 6 In Planta Analysis of the Plastid-Targeting of the IE41Protein

The plasmids 35Ω-IE41-sGFP(S65T), 35Ω-Δ(1-31)IE41-sGFP(S65T),35Ω-Δ(1-59)IE41-sGFP(S65T), 35Ω-Δ(1-99)IE41-sGFP(S65T),35Ω-(6-100)IE41-sGFP(S65T), and 35Ω-(60-100)IE41-sGFP(S65T), and alsothe control plasmids 35Ω-sGFP(S65T) and 35Ω-TP-sGFP(S65T), were digestedwith EcoRI/HindIII in order to recover the expression cassettes.

These cassettes were inserted into the binary plasmid pEL103 (derivedfrom the plasmid pBI121 (AF485783), containing a kanamycin-resistancegene), and the resulting plasmid was used to transform the Agrobacteriumtumefaciens strain C58 by electroporation. The transformed bacteria wereused to transform Arabidopsis WS plants by the “floral dip” technique(The Plant Journal 1998; 16: 735-743). The transgenic plants areselected on the basis of their kanamycin resistance.

In order to analyze the expression of the fusion proteins in thetransgenic plants, the total proteins are extracted from 10 mg of leavesof each of the tested plants, and solubilized in the following buffer:tetrasodium pyro-phosphate (13.4 g/l), Tris-HCl pH 6.8 (50 mM), SDS(1%).

The protein extract is analyzed by SDS-PAGE (12% acrylamide), and byWestern blotting using an anti-GFP antibody (antibody 2A5 (Euromedex)diluted 1/4000 in TBST/5% milk; secondary antibody: alkalinephosphatase-conjugated anti-mouse IgG (Promega) diluted 1/10 000 inTBS-Triton), or the rabbit anti-IE41 polyclonal antibodies described inexample 1 (diluted 1/5000 in TBS-Triton/5% milk; secondary antibody:alkaline phosphatase-conjugated anti-rabbit IgG (Promega) diluted 1/10000 in TBS-Triton buffer).

The results of these analyses are illustrated in FIG. 9;

Legend to FIG. 9:

-   -   A: SDS-PAGE analysis;    -   B: Western blotting with the anti-GFP antibody: the black arrows        indicate the presence of the GFP protein in the fusions        expressed in Arabidopsis;    -   C: Western blotting with an anti-IE41 antibody: the black arrows        indicate the presence of the IE41 protein in the fusions        expressed in Arabidopsis; the white diamond indicates the        position of the natural IE41 protein present in all the        extracts;    -   WT=non-transformed plant;    -   M=molecular weight markers;    -   GFP=plasmid 35Ω-sGFP(S65T);    -   TP GFP=plasmid 35Ω-TP-sGFP(S65T);    -   IE41 GFP=plasmid 35Ω-IE41-sGFP(S65T);    -   Δ(1-59)IE41 GFP=plasmid 35Ω-Δ(1-59)IE41-sGFP(S65T);    -   Δ(1-99)IE41 GFP=plasmid 35Ω-Δ(1-99)IE41-sGFP(S65T);    -   (6-100)IE41 GFP=plasmid 35Ω-(6-100)IE41-sGFP(S65T);    -   (60-100)IE41 GFP=plasmid 35Ω-(60-100)IE41-sGFP(S65T).

These results show that the fusion proteins are expressed in all thetransformed plants.

The subcellular location of the proteins expressed by the variousconstructs was visualized by fluorescence microscopy, as described inexample 5 above.

The results are given in FIG. 10;

Legend of FIG. 10:

-   -   GFP=plasmid 35Ω-sGFP(S65T);    -   TP-RBCS GFP=plasmid 35Ω-TP-sGFP(S65T);    -   IE41 GFP=plasmid 35Ω-IE41-sGFP(S65T);    -   (6-100)IE41 GFP=plasmid 35Ω-(6-100)IE41-sGFP(S65T).

It appears that, under the expression conditions used above, it is theN-terminal region of the IE41 protein (residues 6 to 100) which confersthe greatest specificity of targeting to the plast. In fact, theconstruct (6-100)IE41-GFP, which expresses only this region, allows thesystematic targeting of all the fluorescence to the plasts. On the otherhand, the complete IE41 protein (construct IE41-GFP) induces a lessspecific plastid-targeting. Under these conditions, the IE41 proteinalso appears to be targeted to other intracellular compartments.

1) An intraplastid-targeting polypeptide, characterized in that itcomprises: a domain A consisting of a polypeptide having at least 60%identity, or at least 65% similarity, with one of the polypeptides SEQID NO: 4 or SEQ ID NO: 5; and at least one domain chosen from: a domainB located at the N-terminal end of domain A, and consisting of afragment of one of the polypeptides SEQ ID NO: 1 or SEQ ID NO: 3comprising at least amino acids 49 to 59 of said polypeptide, or else ofa polypeptide having at least 60% identity, or at least 65% similarity,with said fragment; a domain C located at the C-terminal end of domainA, and consisting of a fragment of one of the polypeptides SEQ ID NO: 1or SEQ ID NO: 3 comprising at least amino acids 101 to 111 of saidpolypeptide, or else of a polypeptide having at least 60% identity, orat least 65% similarity, with said fragment. 2) The polypeptide asclaimed in claim 1, characterized in that domain B consists of afragment comprising at least amino acids 39 to 59 of the polypeptidesSEQ ID NO:1 or SEQ ID NO:3, or else of a polypeptide having at least 60%identity, or at least 65% similarity, with said fragment. 3) Thepolypeptide as claimed in either one of claims 1 and 2, characterized inthat domain C consists of a fragment comprising at least amino acids 101to 121 of the polypeptides SEQ ID NO:1 or SEQ ID NO:3, or else of apolypeptide having at least 60% identity, or at least 65% similarity,with said fragment. 4) A chimeric polypeptide, comprising anintraplastid-targeting polypeptide as claimed in any one of claims 1 to3, fused with a heterologous protein. 5) The chimeric polypeptide asclaimed in claim 4, characterized in that the intraplastid-targetingpolypeptide is placed at the N-terminal end of the heterologous protein.6) The use of an intraplastid-targeting polypeptide as claimed in claim1, for the importation of a protein of interest into plasts. 7) The useas claimed in claim 6, characterized in that said intraplastid-targetingpolypeptide is used for the importation of said protein of interest intochloroplasts. 8) A method for importing a protein of interest intoplasts, characterized in that it comprises the expression, in a plantcell containing said plasts, of a chimeric polypeptide resulting fromthe fusion of an intraplastid-targeting polypeptide as claimed in claim1 with said protein of interest. 9) A polynucleotide encoding apolypeptide as claimed in any one of claims 1 to
 5. 10) An expressioncassette comprising a polynucleotide as claimed in claim 9, placed underthe control of sequences for regulating the transcription. 11) Arecombinant vector resulting from the insertion of a polynucleotide asclaimed in claim 9, or of an expression cassette as claimed in claim 10,into a host vector. 12) A transgenic plant transformed with apolynucleotide as claimed in claim 9 or an expression cassette asclaimed in claim 10.